Synopsis

Transmembrane water cycling has long been thought an entirely passive, diffusion-dominated process. Recent studies suggest that an energetically active water cycling (AWC) mechanism, driven by Na⁺-K⁺-ATPase (NKA) pump activity, also occurs in different cell types, including neurons and astrocytes. Here we hypothesize that monitoring AWC could provide a new, more direct physical indicator of neuronal activity, which involves much ion cycling and enhanced NKA activity. By investigating neuronal populations in vitro under resting conditions with spontanotanous activity, and perturbed with tetrodotoxin (TTX), we found TTX simultanously reduces neuronal spiking activity and AWC (by >63%) suggesting AWC directly reflects neuronal activity.

Introduction

Water homeostasis and transport play important roles in brain function, e.g., ion homeostasis, neuronal excitability, cell volume regulation. However, specific mechanisms of water transport across cell membranes in neuronal tissue have not been completely elaborated. In addition to passive water cycling by water diffusing across cell plasma membranes, energetically active water cycling (AWC) also occurs in different cell types^1,2, including neurons and astrocytes^3,4. The AWC is largely driven by cell membrane Na⁺-K⁺-ATPase pump (NKA) activity, where water is co-transported with ion cycling across membrane using secondary active cotransporters to exit and enter the cell (Figure 1)^2,5,6.

Neuronal activity is a highly metabolically demanding process that involves ion transport and recycling: NKA plays an essential role restoring ion gradients⁷. Recently, glutamate transporter activity was found to be strongly coupled to, and regulated by, NKA activity⁸. Here we hypothesize that AWC is postively correlated with neuronal activity, and monitoring AWC could provide a new, more direct physical indicator or measure of neuronal activity in vivo. To test this hypothesis, we investigated neuronal populations (organotypic cortical cultures (OCC) from newborn rat somatosensory cortex) under resting conditions in which spontanotanous activity is observed, and then treated with tetrodotoxin (TTX, a voltage-gated sodium channel (VGSC) blocker), to depress neuronal activity. We simultaneously measure the AWC and neuronal actitivity changes.

Methods

According to our hypothesis, the unidirectional rate constant for steady-state cellular water efflux, k_io, has two components:

k_io = k_io(a) + k_io(p) (1)

where a and p represent active and passive k_io contributions, respectively. For passive exchange, k_io(p) = <A/V>•P_W(p), where <A/V> is the mean cell surface area/volume ratio, and P_W(p) is the diffusion-driven passive membrane water permeability coefficient^1,2. The active rate constant, k_io(a), depends on the cellular metabolic rate of NKA, ^cMR_NKA [e.g., pmol(ATP)/s/cell]. Thus, we rewrite k_io as:

k_io = {x/([H₂O_i]•<V>)}^cMR_NKA + <A/V>•P (p) (2)

where [H₂O_i] is the intracellular water concentration and x is the water stoichiometric multiplier (x = 500 to 1000 per co-transporter (I, IV in Fig. 1). In order to detect ^cMR_NKA changes, k_io(p) changes should be simultaneously estimated.

The kinetics of water exchange in OCC was measured using simultaneous, real-time fluorescence optical imaging and NMR. NMR signals were acquired during perfusion with a paramagnetic agent, Gadoteridol, which enables the determination of k_io, and the mole fraction of intracellular water (p_i), related to the average cell volume (V). Changes in intracellular calcium concentration, [Ca_i²⁺] were used as a proxy for neuronal activity and were monitored by fluorescence. Detailed experimental methods have been reported⁴.

Results

With normal artificial cerebrospinal fluid (ACSF) perfusion, the OCC exhibited spontaneous neuronal activity, demonstrated by the intracellular calcium fluorescence signal (Figure 2A), which exhibited brief (0.5 – 3 s) periods of neuronal excitation separated by ~10 s of very low activity⁹. This baseline spontaneous activity is largely reduced by adding 0.2 µM TTX (Fig. 2A). Interestingly, k_io is also reduced, by 63%, from 2.05 (± 1.67) s^-1 to 0.75 (± 1.13) s^-1 (p = 0.03, Fig. 2B). The mean cell volume also was also reduced during TTX perfusion, with p_i dropping from 0.070 (± 0.018) to 0.057 (± 0.015) (p = 8 x 10^-4).

Assuming P_W(p) and cell shape do not change, the k_io(p) change from baseline to TTX can be estimated from Eq. 2:

{k_io(p,TTX)/k_io(p, baseline)} = {V_baseline/V_TTX}^1/3 = 1/0.95 = 1.05 (3)

In this case, k_io(p) increased by 5%, and, thus, k_io(a) independently decreased by more than 63%.

Discussions and Conclusions

In this study, we found an AWC component in OCC, which is related to neuronal activity. With TTX perturbations, k_io decreased by >63% while cell diameter (d) decreased by 5%. In a previous study with ouabain perturbation, k_io decreased by 45% while d increased by a maximum of 23% (Figure 3)⁴. The two experiments demonstrate that d has no correlation with k_io (Eq. 2), i.e., the active pathway must exist in neuronal tissue.

TTX is frequently used to modulate neuronal spiking activity by blocking VGSC, a Na⁺ influx channel. Here we show that TTX simultaneously reduces neuronal spiking activity and AWC (by at least 63%). A direct consequence of TTX binding would be to reduce Na⁺ influx, and thus decrease NKA activity, which pumps out Na⁺ to maintain the ion’s gradient, and to decrease the NKA activity-driven AWC component. It is probable that VGSC is one of the major III transporters shown in Fig. 1. Since its major activity is generally thought to occur during the action potential, our results suggest that k_io, and specifically k_io(a), directly reflects neuronal activity: ^cMR_NKA increases during the spikes.

Acknowledgements

References

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